Adjusted linear or log mixed effects models quantified WIV and tested potential temporal predictors of continuous or elevated Pro-B concentration. Pearson or Spearman correlations assessed the relationship between averaged repeated biomarker measures and usual citrus intake reported by food frequency questionnaires. The proportion of variance in urinary Pro-B attributable to WIV ranged from 0.69 to 0.74 in unadjusted and adjusted models. Citrus season was a significant predictor of Pro-B in most analyses , while gestational age predicted only non-normalized Pro-B . Moderate correlations (sr of 0.40 to 0.42) were found between reported usual citrus intake and averaged repeated biomarker measurements, which were stronger compared to using a single measurement. Given the high degree of WIV observed in urinary Pro-B, multiple samples per participant are likely needed to assess associations between citrus consumption and health outcomes.Proline betaine (Pro-B) has been identified as a biomarker of dietary citrus intake, yet gaps remain in its validation as a quantitative predictor of intake during various physiological states. This study quantified sources of within-individual variation (WIV) in urinary Pro-B concentration during pregnancy and assessed its correlation with the reported usual intake of citrus fruit and juice. Pro-B concentrations were determined by  Commonly used dietary assessment methods relying on self-report, including the 24-h dietary recall (24HDR) and the food frequency questionnaire (FFQ), are subject to substantial recall bias ,2,3. ThiAlthough dietary biomarkers are not subject to systematic error from recall bias, the use of short-term assessment methods, whether recall- or biomarker-based, to represent long-term usual intake introduces measurement error due to the presence of random, day-to-day variation in dietary intakes within individuals. In addition, many foods and some nutrients  may be episodically consumed; that is, they are not consumed every day (or nearly every day) by most individuals in a study population. For dietary components falling into this category, the aforementioned challenge of within-individual variation (WIV) is exacerbated by the presence of excess zero values in dietary intake data, which further complicates statistical analyses . In areaMeasurement error models developed to account for random WIV in the estimation of a population’s usual dietary intake distributions ,23,24 anCitrus fruits and juices represent one such dietary component that may be of interest to measure for population monitoring and/or nutritional epidemiological studies due to their high content of essential nutrients such as vitamin C, folate, and fiber, as well as other bioactive phytochemicals that may confer health benefits in humans (reviewed in  and 28]28]). SomProline betaine (Pro-B), also known as stachydrine, is a plant osmo-protective compound found in high concentrations in citrus fruits and juices that have emerged as a promising biomarker of citrus intake, given its high abundance in, and relatively high specificity to, these foods ,38,39,40r = 0.29 to r = 0.42) , an internal standard for quantification of metabolites , transferred to 3 mm Bruker  NMR tubes (n = 2 replicates plus 1 phosphate buffer blank sample), and stored briefly at 4 °C. The pH of the samples before data acquisition ranged from 6.83 to 6.85. NMR spectral data were acquired as described previously [Stachydrine (proline betaine) was purchased from Carbosynth LLC . A standard was prepared by combining 11.7 mg of Pro-B with 30 mL of 10 mM phosphate buffer for a final Pro-B concentration of 2.7 mmol/L. eviously  on a BruNMR spectra were manually phase- and baseline-corrected using Chenomx NMR Suite v.8.5 Processor . The Pro-B signature was characterized and saved using Chenomx NMR Suite v.8.5 Spin Simulator, Compound Builder, and Library Manager .®, Siemens, Pharmaforte SingaPore Pte, Ltd., Erlangen, Germany). The supernatant (207 µL) was combined with 23 µL of DSS-d6, as described above. Samples were stored at 4 °C for <24-h before adjusting the pH of each sample to 6.85 ± 0.7 by adding small amounts of HCl and/or NaOH. After transferring samples to 3 mm Bruker NMR tubes, NMR spectral data were collected as described above.Urine specimens collected from MARBLES participants between 2007 and 2014 were stored at −80 °C prior to NMR analysis. Before the current experiment, specimens were subjected to 0–3 (median: 1) freeze-thaw cycles for creating aliquots and/or other purposes. Urine specimens were defrosted on ice, centrifuged at 10 K RCF and 4 °C for 10 min, and then tested for the presence of protein using protein urinalysis strips , the sample-specific lower limit of detection (LOD) was determined by fitting the Pro-B profile in Chenomx to the NMR spectrum at 3.1 and 3.3 ppm (the location of the two most prominent peaks associated with Pro-B) to obtain the highest possible concentration for Pro-B within the signal noise in that spectrum. Values determined to be below the LOD were assigned as 0.5*LOD for use in subsequent steps.After manually correcting the spectral phase and baseline using Chenomx NMR Suite v.8.3 Processor , small water-soluble compounds and metabolites were identified and quantified using Chenomx NMR Suite v.8.3 Profiler . Measured concentrations were adjusted for the dilution attributed to the addition of DSS-d6 and, in the case of two specimens with insufficient sample volume, the addition of Milli-Q Ultrapure HAll analyses were conducted on non-normalized Pro-B as well as Pro-B normalized to creatinine concentration (µmol/mmol creatinine) for comparison. Given the sensitivity limitations of quantification using NMR leading to a substantial number of samples with undetectable Pro-B, and to consider a potentially lower threshold relevant to recent citrus consumption, the urinary Pro-B data were analyzed in two ways. First, samples with non-detectable Pro-B were assigned values of 0.5*LOD, and Pro-B concentration was analyzed as a continuous variable (Approach 1). As a second approach, Pro-B concentrations below a relevant threshold (100 µM or 30 µmol/mmol creatinine) were assumed to be indicative of not having consumed citrus in the last day—whether due to being completely absent or being present in low amounts from other dietary components or from citrus intake in the more distant past—and assigned as zeros (Approach 2). The 100 µM threshold was chosen by selecting the upper end of previously reported urinary Pro-B concentrations  among 26 healthy volunteers after 2 days of abstinence from Pro-B-containing foods . Anothern = 23). Participants reported all foods and beverages consumed in the 24-h prior to spot urine sample collection (n = 20) or coinciding with 24-h urine collections (n = 3). Since full 24HDR was only collected at the beginning of the study before a protocol change, the subsample of pregnancies with available 24HDR all occurred between 2007 and 2008. Reported consumption of any citrus foods or juices on the previous day was analyzed as a dichotomous variable and used to assess agreement with the selected thresholds for urinary Pro-B of 100 µM or 30 µmol/mmol creatinine as indicative of recent citrus consumption. When cases arose in which Pro-B concentration exceeded the threshold but no dietary citrus consumption was reported, items that may contain citrus  were investigated, along with other potential dietary sources of Pro-B. The latter included less commonly consumed food sources or those with a lower Pro-B content than citrus, including gorgonzola cheese, kiwi, pineapple, grapes, seafood, shrimp, mussels, Chinese artichoke (Stachys affinis), rye or whole-wheat products, alfalfa sprouts, capers, and chestnuts [The 24HDR was available for a subsample of participants with corresponding urine samples   was conducted 1–2 times per pregnancy with each participant. The original recall period of the FFQ (one year) was modified by way of written and/or verbal instructions to participants to capture diet from the first or second half of pregnancy. Three items on the FFQ pertained to the frequency and quantity of citrus consumption including (1) oranges or tangerines, (2) grapefruit, and (3) 100% orange or grapefruit juice. The frequency of consumption of each item was converted into frequency per day units. The daily probability (frequency) of consuming any of the three items was determined by the formulaThe citrus season was defined as urine specimen collection occurring between the months of December and May, the approximate time during which citrus fruits are in season in the study area. Gestational age at the time of specimen collection was determined based on the last menstrual period or ultrasound. Demographic and medical information were collected via structured phone interviews, self-administered questionnaires, and/or medical records .Maternal metabolic conditions were categorized as follows: (1) healthy weight with no metabolic conditions, (2) overweight with no metabolic conditions, (3) obesity with no other metabolic conditions, (4) hypertension/preeclampsia without diabetes mellitus, and (5) type 2 diabetes and/or gestational diabetes with or without hypertension/preeclampsia.p < 0.10) with Pro-B in univariate models were considered for inclusion in adjusted analyses. Collinearity of predictors was assessed by testing for significant univariate associations (p < 0.05) in mixed effects models. Urinary creatinine was included as a covariate in non-normalized analyses only. From adjusted models, the residual within- and between-individual components of variance were determined. The proportion of variance attributable to the fixed effects predictors  were obtained by calculating the marginal r-squared of the model using the R statistical package “MuMIn” (version 1.43.17) [n = 247 samples), and (2) randomly selecting a subset with the same number of samples (n = 247).Within-individual predictors of Pro-B concentrations (approach 1) and estimation of variance components: Variance components of urinary Pro-B concentrations treated as a continuous variable were calculated using linear mixed-effects regression models. Within- and between-individual variance components were determined as previously described , includi1.43.17) . Pro-B cWithin-individual predictors of elevated urinary Pro-B and relation with reported daily frequency of citrus consumption: The overall proportion of elevated urinary Pro-B (≥100 µM or ≥30 µmol/mmol creatinine) was described for the whole sample and by the citrus season. Time-dependent and individual-level predictors of elevated Pro-B, along with potential technical/biological confounders, were tested using mixed-effects logistic regression models. Models included a random intercept effect to account for repeated measures, and ten points per axis were used to evaluate the adaptive Gauss-Hermite approximation to the log-likelihood. The same time-dependent, individual, and technical/biological predictors as described above for the continuous Pro-B variable (approach 1) were tested for inclusion in adjusted models using univariate (plus random effects) models. Continuous predictors were scaled as needed to avoid model convergence problems. The assumption of linearity between model predictors and log odds was inspected visually using Loess plots.n is the required number of measurements per individual, Zα is the standardized value for the percentage of times the averaged measured values should fall within the specified limit, wCV is the within-person coefficient of variation, and D0 is the specified degree of error as a percentage of true long-term average excretion. A range of D0 values (5–20%) and a Zα of 1.96 were used in the calculations. For the creatinine-normalized data, the wCV was derived from a mixed effects model after adding a constant of 1 and log transforming the resulting data to avoid negative values in the data used for the wCV calculation.Number of samples required to estimate usual urinary proline betaine of individuals: Within- and between-individual variance components in urinary Pro-B were used to determine the number of repeated measures required to estimate long-term average  Pro-B levels using the formula from Beaton et al. :(2)n=ZαCn is the required number of measurements per individual, r is the correlation level, and 2ws/2bs is the ratio of within- to between-individual variance in Pro-B. Values of r2 ranging from 0.5 to 0.9  were used.To determine the number of samples required to rank individuals on usual Pro-B levels with different levels of accuracy, the following formula from  was usedn = 1, n = 2, n = 3, or n = 4 samples per person. Due to varying numbers of available specimens per participant, sample sizes varied for each of these analyses; thus, correlations using 1, 2, 3, or 4 repeated measures (as available) were repeated on 2 subsets, including (1) individuals with ≥3 samples available (n = 38), and (2) individuals with ≥4 samples available (n = 13).Correlation between urinary proline betaine and reported usual citrus consumption: Mixed effects models as described above for continuous Pro-B or the probability of elevated Pro-B were conducted to determine the association between average FFQ-reported usual citrus consumption  and this biomarker accounting for random day-to-day variation in repeated measures. To address skewness in the data, reported citrus intakes were naturally log-transformed after adding a constant equal to the minimum nonzero intake value multiplied by 0.75 to remove zero values. To determine the level of agreement between repeated biomarker measures and reported usual intake, Pro-B concentrations (approach 1) were averaged within individuals to obtain the best available measure of individual usual Pro-B exposure, and Pearson or Spearman correlations were calculated, as appropriate. To determine the influence of the number of repeated samples per individual on observed correlations, correlation analyses were repeated after subsetting the data to include All analyses were conducted in R Statistical Software version 4.1.0 or 4.3.0 .n = 217), with a maximum of 3 previous thaws (n = 1). Most samples (92.5%) were collected during the second or third trimester of pregnancy  of the NMR spectra, while 2 or more peaks consistent with the Pro-B signal were identified in the remaining samples (Pro-B was not detectable in 22% ( samples . Urinary samples .n = 20) or 24-h urine specimens collected throughout the recall period (n = 3). Using non-normalized Pro-B data, an agreement between citrus consumption reported in the 24HDR and urinary concentrations above 100 µM was 82.6%. Specifically, all 7 recalls indicating citrus consumption had corresponding urinary Pro-B concentrations above the 100 µM threshold, while 4 of the 16 24HDR that did not report citrus consumption had Pro-B concentrations above 100 µM . Covariates in adjusted models included citrus season, gestational age, creatinine, and, in the case of creatinine-normalized data, metabolic conditions. In the creatinine-normalized analysis, a potential multi-collinearity issue was detected between specimen gestational age and one metabolic condition (obesity without the presence of other metabolic conditions), as these were significantly associated (p = 0.046). However, the association between Pro-B and gestational age did not change meaningfully when observations (n = 40) from individuals reporting this condition were excluded, nor were results meaningfully different between adjusted models including either gestational age or metabolic conditions or both. Excluding data from the second pregnancy in cases of multiple pregnancies resulted in similar associations between Pro-B and gestational age.Continuous variable: Two potential sources of within-person variability in urinary Pro-B concentration were tested as predictors in linear mixed effects models. On average, citrus season (December–May) at the time of urine collection was associated with 68% to 72% higher Pro-B in unadjusted models . After ap = 0.08) or adjusted models when considering the non-normalized cutoff (p = 0.04) when considering the creatinine-adjusted threshold  or 0.35 (>30 µmol/mmol creatinine) in the whole sample and varied between specimens collected during the citrus season (December–May) versus those collected between June and November . After ad cutoff . Howeverhreshold .OR = 0.91, CI: 0.78–1.07, p = 0.26) or >30 µmol/mmol creatinine  cutoff was considered.Gestational age at the time of sample collection was not a significant predictor of elevated Pro-B, regardless of whether the >100 μM  the probability of occurrence, and (2) the amount on occurrence days, the latter of which is adjusted for WIV in usual distribution and regression calibration models. Within- and between-individual components of variance were therefore quantified in the portion of urinary Pro-B concentrations falling above the relevant thresholds used in the probability models described above (>100 µM or >30 µmol/mmol creatinine). As expected, WIV and BIV were both substantially reduced compared to these variance components in the continuous data used in approach 1. For non-normalized data above the threshold, variance ratios  were sliwCV for the former. However, when the goal is to rank individuals on Pro-B levels, the required number of samples is similar regardless of normalization to creatinine, given similar WIV:BIV ratios.The within- and between-individual components of variance in urinary Pro-B were used to calculate the number of repeated samples that would be required to (1) estimate true average, long-term  Pro-B levels of individuals, and (2) rank individuals on usual Pro-B levels with varying degrees of accuracy. As shown in The distribution of reported usual citrus intake based on averaged FFQ responses was right skewed, with a median intake of 0.32  citrus servings/day. The distribution of the reported daily frequency of citrus consumption was also right-skewed, with a median frequency of 0.29 .p < 0.0001) and creatinine-normalized  urinary Pro-B on log-transformed scales. When urinary concentrations were averaged among repeated measures within participants, average Pro-B concentration was moderately correlated with reported citrus consumption, with generally stronger correlations found after normalization to creatinine  when using the creatinine-normalized cutoff (proportion = 0.35) but less so when using non-normalized data (proportion = 0.42), and these proportions were less comparable to the median reported daily frequency of 0.29. In unadjusted and adjusted mixed effects models, the frequency of citrus consumption was highly predictive of elevated Pro-B .1H-NMR spectroscopy in repeated spot or 24-h urine specimens, was quantified and potential sources of temporal variation during pregnancy  were identified. In parsing out sources of this variance, citrus season (December–May) was a significant predictor of greater urinary Pro-B concentrations, while gestational age was inversely associated with non-normalized Pro-B concentrations. In linear mixed effects models, a high degree of WIV as a percentage of the total variance in urinary Pro-B was discovered (≥69%) regardless of the normalization method, indicating that multiple samples per participant are likely needed when using this biomarker to assess usual citrus intake or Pro-B exposure. Finally, moderate correlations were found between usual citrus intake reported on a semi-quantitative FFQ and single or averaged repeated urinary Pro-B measurements, with stronger correlations found with repeated measures.This study aimed to inform the use of a urinary biomarker of citrus intake in pregnant women. Here, the magnitude of WIV in urinary Pro-B concentrations, measured by Limited literature exists on the WIV of urinary Pro-B in free-living populations. Wang et al. recently reported intraclass correlation coefficients (ICCs) of relative abundance of candidate dietary biomarkers, including Pro-B, measured in two repeat samples collected 6 months apart, and found a slightly lower proportion of variance from WIV than in the current study (0.56 vs. ≥0.69) . HoweverA number of potential sources of variation may explain the high level of WIV found in urinary Pro-B. First, given the short- to medium-term nature of this biomarker in urine and its strong postprandial response to the intake of citrus products ,39,56,57Citrus season at the time of sample collection was found to be a significant predictor of Pro-B levels above 30 µmol/mmol creatinine and of urinary Pro-B concentration in continuous data analyses. In line with these results, a cohort study of women living in and around Grand Forks, North Dakota found higher reported citrus consumption during the winter and spring seasons . SeasonaA small, statistically significant negative association was found between gestational age and non-normalized urinary Pro-B concentration, suggesting a slight reduction in Pro-B over the course of gestation. In contrast to citrus season, which is assumed to influence Pro-B levels largely through true changes in diet, gestational age could potentially be associated with Pro-B levels either due to dietary changes associated with pregnancy progression or through changes in physiology affecting digestion, metabolism, or urinary excretion dynamics. With regard to diet, some evidence suggests variation in dietary intake of certain food groups ,78 and nWith respect to potential biological effects of gestation on urinary biomarker levels, changes in kidney anatomy and function occurring during pregnancy, e.g., increased glomerular filtration rate and kidney volume and decreased reabsorption of certain substances such as glucose (reviewed in ), could Our study complements and extends upon previously reported data on the correlation between short- to medium-term citrus intake and urinary Pro-B levels ,55,58, a1H-NMR metabolomics approach to measuring Pro-B offers several advantages, including the ability to obtain absolute quantification of multiple compounds using a single experiment, this method is limited in its sensitivity compared to other approaches, such as mass spectrometry, which can detect concentrations in the nanomolar range. The resulting uncertainty in the quantification of Pro-B present in low concentrations, which occurred in a substantial proportion of samples in the present study, may have influenced the variance component estimates reported here. However, we also present a second approach to data analysis that avoids this uncertainty by considering a concentration threshold likely indicative of recent citrus intake. Employing a two-part, probability × amount model, as has previously been conducted in dietary recall analysis to account for the often episodic nature of food consumption [Several limitations should be considered in interpreting the results. First, the use of spot urine specimens collected at the same time of day (first-morning void) may not be ideal for representing citrus consumption over the course of a whole day. Some previous research has observed that most Pro-B excretion occurs in the first 14 h following orange juice consumption ; thus, csumption ,25 may asumption . In the sumption . FinallyThis study also has several strengths. We provide for the first time a quantitative analysis of the within- and between-individual variance components of urinary Pro-B concentration in pregnant women, an understudied population in terms of research on this biomarker. Analyzing up to 5 specimens per person covering all trimesters of pregnancy and seasons allowed us to differentiate random day-to-day variation from two potential sources of systematic WIV in biomarker levels—gestational age and citrus production season—and to test the effect of the number of samples per individual on the correlation with reported usual intake. Collecting >2 repeated measures also may have allowed for better variance component estimation than collecting fewer repeats and/or collecting repeats on a smaller subset of participants, although additional research is needed to determine the ideal timing between and the number of repeated measurements. Another strength was the inclusion of data from spot urine collections along with 24-h specimens, which allowed for the assessment of whether the collection method contributed to variation in the biomarker and for the observation of a higher degree of WIV in Pro-B among spot urine samples. In the context of large epidemiological studies, using spot urine samples carries the advantage of having a smaller participant burden relative to 24-h collections; thus, the results presented here can inform future studies aiming to use this measure to assess usual citrus intake in free-living populations.Some additional research is warranted before relying on Pro-B as a quantitative measure of citrus intake, whether acute or usual, for the purposes of replacing, validating, or correcting for measurement errors in dietary recall instruments. Notably, recently developed calibration equations that successfully predicted mean total citrus intake over 4 consecutive days based on a single first void urine specimen in an Irish population ,58 are pA high degree of WIV in urinary Pro-B concentrations was observed among pregnant women in a Northern Californian cohort. In this area of high local citrus production, citrus season accounted for a small, statistically significant portion of the total variance in Pro-B (3%). The contribution of gestational age to WIV of Pro-B was less clear, as a weak negative association was observed with non-normalized Pro-B only. Single or averaged repeated urinary Pro-B concentrations (1–5 samples per person) were moderately correlated with usual citrus intake as reported by FFQ. Collecting ≥2 urine specimens per individual may strengthen this correlation and more closely represent usual intake, but more research with a larger sample size of individuals with ≥3 repeated measurements is needed to confirm the additional benefit of a greater number of repeats. Usual intake/exposure models based on the decomposition of the total variance into WIV and BIV are likely necessary to represent usual citrus intake/Pro-B exposure more accurately for the purpose of characterizing population distributions or epidemiological associations. NMR spectroscopy is a convenient method for quantifying Pro-B in urine, particularly if a simultaneous measurement of multiple biomarkers is desired, but may require the use of relevant cutoffs and 2-part (probability × amount) models for accurate analysis given the limited sensitivity of the method. Given the often-episodic nature of citrus fruit consumption and the high degree of WIV in biomarker values reported here, multiple urine specimens per individual should be collected in dietary studies aiming to quantify usual citrus intake and/or Pro-B exposure.


In a parallel experiment, it was determined whether ergothioneine (EGT) contained in Rice-koji displayed similar effects. Anxiety and nociception were assessed behaviorally using multiple procedures. c-Fos and FosB immunoreactivities were quantified to assess the effect of both treatments on neural responses in the paraventricular nucleus of the hypothalamus (PVN), nucleus raphe magnus (NRM), and lumbar spinal dorsal horn (DH). FST increased anxiety- and pain-like behaviors in the hindpaw. Rice-koji or EGT significantly prevented these behaviors after FST. In the absence of formalin, both treatments prevented decreased FosB expressions in the PVN after FST, while no effect was seen in the NRM and DH. In the presence of formalin, both treatments prevented changes in c-Fos and FosB expressions in all areas in FST mice. Further, in vitro experiments using SH-SY5Y cells were conducted. Rice-koji and EGT did not affect cell viability but changed the level of brain-derived neurotrophic factor. In conclusion, Rice-koji could reduce anxiety and pain associated with psychophysical stress, possibly mediated by the modulatory effects of EGT on neural functions in the brain.This study determined the effect of daily administration of Rice- The management of mental illness and chronic pain induced by psychological stress could rely on the use of drugs. However, the effectiveness of drug treatments is inconsistent. Thus, better ways to cope with psychological stress daily are important for preventing anxiety and chronic pain because psychological stress is less avoidable in our life. Lifestyle medical approaches such as nutrition inventions have been shown to control psychological stress-related disorders .koji, made from Aspergillus oryzae (yellow koji mold), is a readily accessible contributor to nutrition and has been used as a source of rice-fermented food [koji are due to the abundance of substances contained in it that have the potential to regulate bodily function [Aspergillus oryzae, could prevent psychophysical distress and nociception [koji is a starter in the production of sake [koji on psychological stress are unclear. Lately, consumers have been preferring natural foods, including rice-fermented foods, because of the increased interest in safer diets in Japan ,3. Rice-ted food ,5,6. Thefunction . Our preiception ,9. Sake  of sake ; howeverkoji can regulate body functions [koji, is a derivative of an amino acid that is considered to provide health benefits [Aspergillus oryzae is an EGT-rich food [Several bioactive compounds contained in Rice-unctions . For exabenefits . Fermentich food , while Eich food  and painich food ,16 behavich food .koji extracts (Rice-koji) can prevent anxiety- and pain-like behaviors under psychophysical stress conditions. In a parallel study, the effects of EGT on these responses were evaluated. These experiments could elucidate the possible molecular basis for the modulatory effects of Rice-koji on psychophysical stress responses in part. A paradigm of repeated forced swim stress (FST) was conducted to develop a psychophysical stress model in mice [koji and EGT on cognitive functions were also determined because psychological stress can cause cognitive impairments [koji and EGT on pain-like behaviors under FST conditions was determined. A report revealed the inhibitory roles of EGT on neuropathic pain-like behaviors [koji and EGT have inhibitory effects on nociception associated with psychological stress conditions. The first aim of this study was to determine whether Rice- in mice . Multipl in mice . Furtherairments . The novairments ,21. Furtehaviors ; howeverkoji and EGT on neural responses indicated by c-Fos and FosB immunoreactivities, markers for neural activity, in the brain. This study focused on several areas, including the paraventricular nucleus in the hypothalamus (PVN), nucleus raphe magnus (NRM), and lumbar spinal dorsal horn (DH) [koji and EGT. The second aim of this study was to determine the effect of Rice-orn (DH) . The PVNorn (DH) ,24, and orn (DH) ,25,26, aorn (DH) ,27,28. Norn (DH) ,30. Prevorn (DH) . Howeverkoji and EGT on neural responses using human neuroblastoma cells. SH-SY5Y cells, so-called human neuron-like cells, are used as an in vitro model for neural disorders [koji itself has any negative effects on cell survival. Next, the level of brain-derived neurotrophic factor (BDNF) of the SH-SY5Y cells was assessed in the presence of Rice-koji or EGT. Because changes in BDNF signaling can cause psychological illness and chronic pain [koji and EGT exert modulatory effects on neural functions at the cellular level.This study also determined the potential effects of Rice-isorders ,32,33,34nic pain , measurekoji could prevent anxiety and nociceptive responses associated with psychophysical stress through the modulation of neural responses in the brain due to the actions of EGT.These notions indicate that Rice-koji was provided by the local sake brewery  and dissolved in PBS buffer at 0.5 g/mL concentration. Then, the supernatants after centrifugation  were filtered through a microfilter with a pore size of 0.22 µm and were used as a high dose of Rice-koji extracts (Rice-koji). The high dose of Rice-koji (High dose) was further diluted with PBS ×0.1, which was used as a low dose of Rice-koji . Rice-koji was stored at −20 °C until use. EGT  was diluted in saline, and 0.1 mg/kg and 1 mg/kg EGT were employed [Rice-employed .koji (High dose: 0.5 g/mL) was diluted with 50% acetonitrile solution and filtered through a 0.2 μm membrane filter. The quantitative value of the sample was obtained by the internal standard method. EGT was quantified by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Prominence UFLC  was used as the liquid chromatography instrument, and a ZORBAX Rx-SIL  column was used at 40 °C. The injection volume was 5 µL. Mass spectrometry was performed using a 4500 Qtrap  with electrospray ionization.Rice-We employed 200 male C57BL/6J mice. All animal experimental protocols were reviewed and approved by the Intramural Animal Care and Veterinary Science Committee of Niigata University (permit number: SA00611) and performed in accordance with the Guiding Principles for Care and Use of Laboratory Animals . Male mice  were employed. Mice were kept in cages (4 mice per cage) made of transparent acrylic (30 × 20 × 15 cm) with free access to food and water. Cages were kept in a temperature- and humidity-controlled environment with a 12:12 light/dark cycle (light phase: 7:00–19:00).This study employed a modified psychophysical model of rodents, which displayed a sustained behavior of despair for 1–3 weeks . A repeakoji (Low or High doses) and EGT (0.1 or 1.0 mg/kg) were administered orally at 0.2 mL/mouse through a stomach tube 30 min and 6 h after each Sham or FST experiment on Days 1, 2, and 3 , the social interaction (SI), open field (OF), novel object recognition (OR), and hindpaw formalin tests were conducted. In Experiment 2 , the elevated plus maze, dark and light box, and pain-like behavioral tests evoked by heat and mechanical pressure stimulation were conducted. In each experiment, Sham and FST mice were divided into 5 groups as follows: vehicle -, Rice-koji (Low and High dose)-, and EGT (0.1 and 1.0 mg/kg)-treated groups. For the immunohistochemical (IHC) experiments, several mice were randomly selected and divided into two groups with or without formalin injection into the left hindpaw on Day 5. The experimenters who conducted the following behaviors and IHC experiments were blind to treatments. The number of animals employed in each experiment is present in the figure legends.Mice were separated into two experiments: Experiment 1 and Experiment 2 . In Expekoji and EGT on the conditions of psychological distress and recognition  was recorded. Immobility behaviors were when the mouse remained afloat in the water without struggling and only made the movements required to keep its head just above the water. The experimenter was blinded to all treatments.2.Open field (OF) testThe OF test was employed to measure locomotor activity on Day −1 and Day 3. Mice were placed in a clear acrylic cage with black frosting Plexiglas floor (45 × 45 × 30 cm) for 5 min. The total movement distances and the time spent in the center zone area were measured using digital counters with infrared sensors . On Day 3, the OF test was conducted 5–6 h after the FST conditioning. The experimenter was blinded to all treatments.3.Social interaction (SI) testThe SI was conducted on Day 0 and Day 4. Mice were put in the center of a clear acrylic cage with a black frosting Plexiglas floor that served as an open field area (45 × 45 × 30 cm). The wide area was divided into two interest zones: the interaction zone (IZ) and corner zone (CZ). The SI test consisted of two sessions (2.5 min each). For the first session, mice were placed in the open field without the male stranger mouse (Institute of Cancer Research (ICR) mouse, 30–35 g) in the IZ. Next, 1 min later, the ICR mouse was placed in the middle of IZ inside the perforated acrylic enclosure. In both sessions, the time spent in the IZ was recorded using digital counters with infrared sensors . The experimenter was blinded to all treatments.4.Elevated plus maze (EPM) testOn Day 0 and Day 4, a mouse was placed in the center of a maze that consisted of 2 opened and 2 closed arms. The maze was raised around 50 cm above the ground . The duration of time spent in open arms was recorded for 5 min. The experimenter was blinded to all treatments.5.Dark and light box (DL) testOn Day 0 and Day 4, a mouse was placed in a dark and light box with two compartments, one a light chamber and the other a dark chamber, which were joined by an opening. Mice were initially placed in the light chamber, and the behavior of the mouse was observed for 15 min. The time spent in each chamber was measured by digital counters with infrared sensors . The DL test was conducted 2 h after the EPM tests. The experimenter was blinded to all treatments.6.Novel object recognition (OR) testOR is a measure of recognition memory and primarily relies on the natural tendency of mice to spend more time exploring a novel object than a previously encountered one ,21. The koji and EGT on the pain-like behaviors evoked by three different modalities, heat, mechanical pressure, and formalin, were measured in Sham and FST miceEffects of Rice-Hindpaw heat sensitivityHeat withdrawal latency was measured on Day −1 and Day 4  paw-pre3.Hindpaw formalin testThe hindpaw formalin test was conducted on Day 5 to assess the nocifensive behaviors of mice. Two microliters of 2.5% formalin was injected subcutaneously into the left hindpaw, and then mice were returned to the cage for a 45 min observation period. A stopwatch was used for 15 consecutive 3 min intervals to record the time spent on licking behaviors at the injected hindpaw. Licking behavior responses were divided into 2 phases—early phase (0–9 min) and late phase (9–45 min)—to analyze the results. The experimenter was blinded to all treatments.On Day 5, 5–10 animals were randomly selected in each treatment group and subjected to c-Fos and FosB immunohistochemical experiments 24 h after the last behavioral tests conducted on Day 4 . Animals2 at 37 °C. For the neuronal induction of SH-SY5Y cell lines, the culture medium was replaced with a differentiation medium containing 10 μM of all-trans retinoic acid  in the presence of Rice-koji extracts (10 or 500 µg in 1 mL PBS), EGT (10 or 100 µg in 1 mL PBS), or PBS alone. The differentiation medium was replaced every 5 days over 4 weeks of incubation [SH-SY5Y cells are often employed as the in vitro models for anxiety  and paincubation .2 at 37 °C. Subsequently, the same volume of MTT extraction solution (10% SDS and 0.01 N HCl) was added to the culture medium, and the absorbance at 560 nm was measured after 16 h at room temperature.An MTT assay was conducted. MTT solution was added at a final concentration of 500 μg/mL to the SH-SY5Y cells cultured in 6-well plates 28 days after the induction of differentiation and incubated for 3 h in a humidified atmosphere of 5% COThe level of brain-derived neurotrophic factor (BDNF) was measured using the Mature BDNF ELISA Kit . The experimental procedure followed the manual accompanying the kit. In brief, the culture medium of SH-SY5Y cells at 28 days after induction of differentiation was added to the ELISA plate and incubated at 25 °C for 2 h. After washing, subsequently, a biotin-conjugated specific antibody was incubated at 25 °C for 1 h to bind the captured BDNF. Then, peroxidase-conjugated streptavidin solution was added and incubated at 25 °C for 30 min. Unbound conjugates were removed by subsequent washing. Plates were incubated with TMB , and the absorbance at 450 nm was measured. All samples were assayed in triplicate.koji- and EGT-treated groups. Time spent on formalin-evoked licking behaviors for 45 min observation was compared with formalin-evoked c-Fos and FosB expressions in the PVN, NRM, and DH. In the case of the DH, the numbers of c-Fos- and FosB-positive cells in laminae I–II and III–V were combined. The sample size in each treatment group is shown in the figure legend. A statistical level of p < 0.05 was considered significant.Statistical analyses for the behavioral, IHC, MTT assay, and ELISA experiments were performed using SPSS Statistics . Data of behavioral, MTT assay, and ELISA experiments were assessed by analysis of variance (ANOVA), while those of IHC were analyzed by two-way ANOVA . The results are presented as the average ± standard deviation (SD). Post hoc tests comprising individual comparisons were performed using the Bonferroni test. Spearman’s test was conducted to assess the correlation between the number of nociceptive neural responses in the brain versus pain-like behaviors and was separately conducted using the results obtained from Rice-koji extract (Rice-koji) by LC-MS/MS to measure how much EGT was contained. The result revealed that 1.0 g of Rice-koji contained 13.0 μg of EGT. The dose of EGT contained in Rice-koji (High dose), which was administered to each mouse, was equivalent to that of 0.1 mg/kg EGT. The data of the chromatogram are presented in We analyzed our Rice-koji  and EGT  on Day 2 and Day 3 was significantly smaller than that in those treated with saline  and time spent in the center area (p < 0.01) on Day 3 were significantly decreased compared with on Day −1. In Rice-koji (High dose)- and EGT (0.1 and 1.0 mg/kg)-treated groups, total movement distance (p < 0.01) and time spent in the center area (p < 0.01) were significantly greater than in the saline-treated group.In FST mice treated with saline, total movement distance , time spent in the IZ in the presence (+) of the ICR mouse was significantly shorter than in the absence (−) of the ICR mouse  and EGT (0.1 and 1.0 mg/kg) was significantly longer than that spent by FST mice with saline  by Sham mice was similar between treatment groups in the absence (−) and presence (+) of the ICR mouse p > 0.1, F,G. In Fkoji, and EGT (p < 0.01) and Rice-koji  groups on Day 4 was significantly decreased compared with on Day 0 (koji (High doses) and EGT (0.1 and 1.0 mg/kg) was significantly longer than that spent by the saline group , while EGT .In Sham mice treated with saline and Rice-p < 0.01) and Rice-koji  (koji (High dose) and EGT (0.1 mg/kg) on Day 4 was similar to that on Day 0  and FST (p > 0.1) mice. In Test 2, the time spent on the novel object (in “the B area”) by Sham mice treated with saline, Rice-koji, and EGT was significantly greater than time spent in “the A area” , time spent on the novel object (in “the B area”) was similar to that spent in “the A area”  and EGT  was significantly greater than that spent in “the A area” , Rice-koji , and EGT , the withdrawal latency on Day 4 was significantly decreased compared with that on Day −1. However, on Day 4, the withdrawal latency in FST mice treated with Rice-koji (High dose) and EGT (1.0 mg/kg) was significantly longer than in those treated with saline , Rice-koji (p < 0.01), and EGT (p < 0.01), the withdrawal threshold on Day 4 was significantly decreased compared with that on Day 0, with no significant difference between treatment groups . Rice-koji and EGT did not change such nocifensive behaviors in Sham and FST mice  and EGT (0.1 and 1.0 mg/kg) was significantly shorter than in those treated with saline. .In the late phase, time spent on licking behaviors by saline-treated FST mice was significantly longer than that spent by Sham mice (p > 0.1), and Rice-koji (p > 0.1) and EGT (p > 0.1) did not change the number of c-Fos-positive cells in Sham and FST mice. In saline-treated groups (p < 0.01). Further, in the FST group, the number of FosB-positive cells was significantly greater in EGT (0.1 and 1.0 mg/kg)-treated mice than in saline-treated mice (p < 0.05).In the absence of formalin injection (p < 0.01), Rice-koji- (p < 0.01), and EGT-treated (p < 0.01) groups was greater than in the same treatment groups without formalin injection. In the saline-treated group, the number of c-Fos-positive cells in FST mice was significantly smaller than that in Sham mice (p < 0.01), whereas the number of c-Fos-positive cells in EGT -treated mice was significantly greater than in saline-treated FST mice. The number of FosB-positive cells in FST mice treated with Rice-koji  and EGT  was significantly greater than in saline-treated FST mice.In the presence of formalin injection A, the nup > 0.1) and FosB-positive (p > 0.1) cells in saline-treated FST mice was similar to that in Sham mice. Rice-koji (p > 0.1) and EGT (p > 0.1) did not alter the number of c-Fos and FosB expressions in Sham and FST mice.In the absence of formalin injection B, the nukoji, and EGT was significantly greater than in the same treatment groups without formalin injection in Sham and FST mice (p < 0.01). The number of c-Fos-positive cells in FST mice treated with Rice-koji (High dose) and EGT (1.0 mg/kg) was significantly smaller than in the saline group ; however, the number of FosB-positive cells in EGT (1.0 mg/kg)-treated FST mice was significantly smaller than in the saline group .In the absence of formalin injection, FST alone did not change c-Fos (p < 0.01) and III–V (p < 0.01) in saline-treated FST mice was significantly greater than in Sham mice (p < 0.01) and FST (p < 0.01) mice treated with Rice-koji (High dose) and EGT (1.0 mg/kg) was significantly smaller than that in those treated with saline - and EGT -treated groups was smaller than in the saline-treated group in laminae I–II. However, in laminae III–V, FosB expressions were not changed by Rice-koji and EGT (p > 0.1).The number of formalin-evoked FosB-positive cells in laminae I–II in saline-treated FST mice was greater than in Sham mice  and NRM  was significantly correlated with pain-like behaviors in the Rice-koji and EGT treatment groups (p < 0.0001) but not in the NRM . Further, formalin-evoked c-Fos expressions in the PVN  were negatively correlated with pain-like behaviors; however, FosB expressions were not .The c-Fos expression in the DH  was smaller than in those treated with PBS on Day 28 (p < 0.01). The level of BDNF in SH-SY5Y cells treated with EGT at doses of 10 and 100 µg/mL was greater (p < 0.01) and smaller (p < 0.01) than in those treated with PBS, respectively  on stress-induced anxiety- and pain-like behaviors, consistent with several previous findings. Oral administration of EGT inhibited depression-like behaviors identified by FST and tail suspension tests  and coulkoji on anxiety- and pain-like behaviors because EGT is one of the ingredients contained in rice-fermented foods and is a naturally occurring food-derived antioxidant that can regulate neural functions [koji, and the actual dose of EGT contained in 0.1 mg/kg EGT was the equivalent dose of EGT contained in the High dose of Rice-koji. It is noteworthy that daily administration of the High dose of Rice-koji or EGT at a dosage of 0.1 mg/kg decreased anxiety- and pain-like behaviors in FST mice. These findings suggest that the inhibitory effects of Rice-koji on psychological stress might be mediated through the actions of EGT.These results further allow us to elucidate a possible molecular basis for the preventive effects of Rice-unctions . Oxidatiunctions . Our LC-This study employed multiple behavioral procedures to evaluate anxiety- and pain-like responses in mice subjected to FST. FST is a traditional procedure used to investigate new antidepressant drugs first described by Porsolt et al. . Severalkoji were determined in FST mice, and a high dose of Rice-koji prevented anxiety-like behaviors, identified in all behavioral procedures. However, the effect of a low dose of Rice-koji seemed to be less, except for in the OF test. These findings indicated that sensitivity to the detection of anxiety-like behaviors was different between the methodologies for the assessments of behavior. However, the functional significance of the different results between behavior tests remains unclear.Anxiolytic effects of Rice-koji and EGT exerted less of an effect on anxiety-like behaviors, except in the results of the DL test. Of note, EGT increased the time spent in the light chamber on Day 4  behavioral test revealed that time spent at the novel object  and EGT (0.1 and 1.0 mg/kg) increased the time spent at the novel object (“the B area”) compared with that spent at the familiar one (“the A area”). These findings indicated that Rice-koji and EGT might protect FST-induced dysfunction of recognition, while these treatments exerted less of an effect in Sham mice. A clinical study supports our findings, in part, stating that the concentration of EGT in blood serum was reduced in older people with cognitive impairment [We also determined whether Rice-pairment .koji and EGT prevented thermal- and formalin- but not mechanical-stimulation-, evoked nocifensive behaviors in FST mice. These results are consistent with our previous report on the inhibitory roles of another rice-fermented food, sake lees, which could prevent formalin but not mechanical nocifensive behaviors in the hindpaw in FST mice [koji nor sake lees [In accordance with the effects on anxiety-like behaviors, Rice-FST mice . Althougake lees  inhibitekoji exerted less of an effect on nocifensive behaviors  and EGT reduced formalin-evoked c-Fos expressions in the NRM (koji and EGT on FosB expressions was smaller than that on c-Fos expressions. Rice-koji and EGT decreased FosB expressions in laminae I–II but not in laminae III–V. The positive correlation of both markers in the DH and that of c-Fos expressions in the NRM evoked by formalin injection supported the notions that nociceptive neural activities in the DH and NRM are significantly correlated with pain-like behaviors [In FST mice, Rice- the NRM  and in b the NRM . These rehaviors ,64.koji and EGT can regulate c-Fos and FosB expressions in mice is beyond the scope of this study, our in vitro results demonstrated that Rice-koji and EGT could have some influences on neural function at the cellular level. Indeed, our ELISA analysis revealed that Rice-koji decreased while EGT increased and decreased the BDNF level of the SH-SY5Y cells depending on the concentrations. These findings might offer the possible mechanisms by which Rice-koji and EGT affect cellular functions, which could regulate neural responses in the brain. However, the linkage of bidirectional changes in the BDNF level in the SH-SY5Y cells to the regulation of anxiety- and pain-like responses remains unclear because either increases or decreases in the level of BDNF associated with psychological stress could be dependent on the areas in the brain. Psychological stress increased the BDNF level in the anterior cingulate cortex and insular cortex [Although elucidation on the precise mechanism of how Rice-r cortex  and decrr cortex  and hippr cortex .koji and EGT on cell viability. It is, however, unlikely because our MTT assay displayed no reduction of it. At this point, it is unclear how the current in vitro results could be connected with the results of the in vivo experiment. However, emphasis is placed on the notion that both treatments exerted a significant effect on the functional activity at the cellular level at least [Further, one might consider that decreases in the BDNF level could be due to the inhibitory effect of Rice-at least .koji could alleviate anxiety- and pain-like responses, with changes in neural responses in several areas of the brain under psychophysical stress conditions, and such anti-stress effects might be due to the action of EGT which is contained in Rice-koji. This is the first report on the beneficial effects of Rice-koji on psychological stress conditions despite the evidence of its traditional use.Daily administration of Rice-


Catha edulis) is an evergreen shrub whose buds and leaves give a state of delight and euphoria when chewed. Cathinone, an amphetamine-like stimulant that is among the active ingredients in khat, is able to downregulate glutamate transporter subtype I (GLT-1). Neurobehavioral dysfunctions such as altered locomotor activity, anorexia, and nociception have been observed in animals exposed to cathinone. Interestingly, treatment with a β-lactam antibiotic such as ceftriaxone, which upregulates GLT-1, normalizes cathinone-induced conditioned place preference, and alters repetitive movements in rats. However, little is known about the role of the glutamatergic system in memory dysfunction and anxiety-like behaviors in mice exposed to khat. We found here that clavulanic acid, a β-lactam-containing compound and GLT-1 upregulator, would modulate the neurobehavioral changes, including memory impairment and anxiety-like behaviors, associated with repeated exposure of mice to khat. Our data supported that clavulanic acid could improve memory impairment and anxiety-like behaviors through upregulating GLT-1 in the nucleus accumbens (NAc), an effect abolished with a selective GLT-1 blocker. This upregulation was associated with restored glutamate/cystine antiporter expression in the NAc using a Western blotting assay. Cathine and cathinone were identified in khat extract using the gas chromatography technique. Our work provides preclinical insight into the efficacy of β-lactam-containing compounds for the attenuation of neurobehavioral changes induced by khat exposure.Khat ( Catha edulis) is a seedless plant of the family Celastraceae whose leaves have an aromatic odor and astringent and relatively sweet taste; it can grow in various climates and soils and is cultivated as a bush or a small tree [Khat  ,15. UndeEAAT1  is highly expressed in the cerebellum. EAAT2  is the primary glutamate transporter in the forebrain; it is abundant in astrocytes but expressed only to a limited extent in presynaptic nerve terminals . AnotherAs mentioned previously, glutamate is rapidly eliminated from the extracellular space under normal conditions through binding and uptake by cell-surface transporters. This rapid removal is essential to ensure controlled and efficient cell-to-cell communication during homeostasis. Astrocytes are primarily responsible for glutamate uptake and catabolize and convert it further into glutamine, then deliver the glutamine back to presynaptic neurons . Given tImportantly, glutamate pathways are impacted in substance use disorders. Five weeks of ethanol drinking reduces the protein expression of GLT-1 and xCT in central reward brain regions, including the nucleus accumbens (NAc), amygdala, and hippocampus ,28, and Synthetic cathinone compounds, commonly referred to as “bath salts”, are a popular class of new psychoactive substances. Repeated exposure to synthetic cathinone, 3,4-methylenedioxypyrovalerone (MDPV), followed by withdrawal, has been observed to reduce GLT-1 protein expression in the NAc of rats on day 5 and day 10 later . Accordiβ-lactam antibiotics have demonstrated the ability to attenuate ethanol-, cocaine-, nicotine-, and hydrocodone-seeking behaviors, at least in part by increasing the expression of GLT-1 protein expression in mesocorticolimbic brain regions ,31,34,40The average body weight of the mice in each group was measured throughout the study period. Using statistical analysis by a two-way ANOVA, we did not reveal any significant differences between the groups, either at baseline or post-treatment for time F  = 1.867, for treatment F  = 1.353, and for interaction F  = 0.1730) .Locomotor activity was monitored for all groups during the EPM and NORT experiments.t-tests. No statistically significant difference was observed for any of the groups  and the DHK-CA-Khat group (p < 0.01) using paired t-tests . For the Control and CA-Khat groups, no significant difference was observed (p < 0.05). However, these differences did not remain significant after applying Tukey’s post hoc test.We found statistically significant differences in the pre- and post-intervention closed-arm time ratios within the Khat group  and the Khat-CA-DHK group (p < 0.01) (p < 0.05), but the difference did not remain significant after applying Tukey’s post hoc test.Using paired  < 0.01) B  and Khat-CA-DHK groups only (p < 0.01) (p < 0.001). Post hoc comparisons using Tukey’s test indicated the total time spent in closed arms to be higher in the Khat and Khat-CA-DHK groups as compared with the Control and Khat-CA groups  A  and the Khat-CA-DHK group (p < 0.01) (p = 0.0588) using a one-way ANOVA.The total time mice spent in open arms before and after intervention was compared using paired  < 0.01) B  and the Khat-CA-DHK group (p < 0.05) (p < 0.001) using a one-way ANOVA, but not in the baseline phase  = 1.073). After applying post hoc comparisons using Tukey’s multiple comparison test, we reported a statistically significant lower discrimination index in the Khat and Khat-CA-DHK groups as compared with the Control and between Khat and Khat-CA groups (Using paired  < 0.05) A (ControA groups A.p < 0.001) and the Khat-CA-DHK group (p < 0.05) were found using paired t-tests (p < 0.001), but not in the baseline phase  = 1.696). Statistically significant differences were found between the Control/Khat, Khat/Khat-CA, and Khat-CA/Khat-CA-DHK groups using post hoc Tukey’s comparisons test  using a one-way ANOVA. Subsequent post-hoc analysis using Tukey’s multiple comparison test, we reported significant differences to exist between the Khat group and those that received khat and clavulanic acid (p < 0.01), as well as between the Control group and the Khat group (p < 0.01)  = 9.871,  < 0.01) .p = 0.0005) using a one-way ANOVA. After applying post hoc analysis with Tukey’s multiple comparison test, we identified significant differences between the Khat group and those that received khat and clavulanic acid (p < 0.01), as well as between the Control group and the Khat group (p < 0.01)  = 13.13,  < 0.01) .p = 0.2156)  = 1.703,  0.2156) .GC-MS analysis was used to quantify the two major psychostimulant active ingredients, cathine and cathinone, in the khat extract used in the present study. This detection was performed with reference to external standards of different known concentrations of cathine and cathinone A. The peThe use of khat extract is reported to have both physical effects, such as weight loss, and behavioral effects, such as increased locomotor activity and anxiety. These effects may be attributed, at least in part, to impaired uptake of the excitatory neurotransmitter glutamate. In the current work, we hypothesized that the β-lactam compound clavulanic acid, which upregulates the glutamate transporter GLT-1, could modulate the neurotoxic and neurobehavioral changes associated with repeated khat extract exposure in mice.Regarding physical effects, a previous study found body mass index to be lower in khat users as compared to controls, although lean body mass was not affected . In the Khat extract has been demonstrated to increase locomotor activity. However, in the present study, repeated administration of khat to C57BL/6 mice did not affect locomotor activity . Kimani et al. performed daily i.p. injections on mice with khat for 17 days  and founv/v, 2 g/kg) and clavulanic acid  were given simultaneously by oral gavage for ten days, after which EPM tests showed all groups receiving clavulanic acid to enter open arms more often and spend more time in them, supporting a positive effect of clavulanic acid on drug-induced anxiety behaviors [Regarding behavioral effects, the EPM test was used to evaluate anxiety-related behavior in mice receiving four different treatments, with the ratio of time spent in closed vs. open arms taken as indicative of anxiety. The results showed repeated administration of khat extract to induce anxiety-like behaviors, an effect that was abolished with seven days of clavulanic acid treatment . Additionally, the effect of clavulanic acid was prevented by additional treatment with DHK, suggesting GLT-1 to be involved in this attenuation. Bedada et al. showed a similar increase in anxiety behaviors among mice receiving 200 mg/kg of khat extract , and anoehaviors .We also used the NORT to assess memory defects in mice receiving repeated administration of khat extract, in which test greater discrimination index and more time spent in the novel object zone are considered to indicate improved memory. Statistically significant changes in discrimination ratio and time spent with the novel object were observed in the Khat group after 17 days of exposure. This memory dysfunction was rescued by a 7-day treatment with clavulanic acid, which effect was, in turn, inhibited by DHK. These findings support that repeated administration of khat impairs memory recognition compared with the baseline state and that this effect may be mediated through GLT-1, which is upregulated by clavulanic acid. In a similar study, Limenie et al. investigated the effects of sub-chronic khat extract exposure on learning and memory in Swiss albino rats . The autWe next evaluated the effect of repeated exposure to khat extract with/without clavulanic acid treatment on GLT-1 expression in the mouse NAc. Repeated administration of khat extract resulted in downregulation of GLT-1, while clavulanic acid treatment restored its expression. The observed khat-induced downregulation of GLT-1 is consistent with previous research showing that withdrawal from chronic exposure to MDPV, a synthetic cathinone, reduces GLT-1 expression in the NAc . MoreoveWe further investigated the effect of repeated khat exposure and clavulanic acid treatment on xCT expression in the mouse NAc. The results showed repeated khat administration to decrease xCT expression and clavulanic acid treatment to reverse this effect. These findings concur with a number of preclinical studies that demonstrated xCT expression in the NAc to be decreased in animals exposed to drugs of abuse such as methamphetamine, cocaine and hydrocodone, and furthermore showed β-lactam compounds to decrease behavioral symptoms and restore xCT expression ,33,53. ITo further characterize the effects of khat extract on astroglial glutamate transporters, we analyzed protein expression of the GLAST glutamate-aspartate transporter in mice treated with khat extract with/without clavulanic acid treatment. This revealed no significant difference between any of the experimental groups. Prior studies have likewise reported that exposure to hydrocodone, cocaine, or methamphetamine does not alter GLAST expression in the mesocorticolimbic system ,33,53. Tv/v) for 72 h at room temperature with continuous shaking. The supernatant was filtered through filter paper (0.45 µm) twice, and the obtained extract was allowed to dry at room temperature. It was then weighed, and the percent yield was determined (10%). The extract was finally refrigerated at 4 °C in dark bottles until further use [Khat was provided by Jazan University, Jazan, Saudi Arabia. It was prepared and extracted using a specific apparatus according to a procedure previously described by Richardson and Harborne . Brieflyther use .® 20 detergent (TBST), and rabbit primary antibodies  were purchased from ABclonal, Wuhan, China. The housekeeping antibody, rabbit anti-β-actin, was also purchased from ABclonal, Wuhan, China.Clavulanic acid , dihydrokainic acid (DHK) , and physiological normal saline (0.9%) were prepared daily in the lab. For Western blots, 8–15% SDS-PAGE gels, 3% non-fat milk in Tris-buffered saline with 0.1% TweenForty male C57BL/6 mice aged 5–8 weeks and weighing 20–25 g were housed in standard group cages and kept under a 12 h light/dark cycle. The mice were obtained from the Experimental Animal Care Centre, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia. The mice were kept in a room with a humidity of 40 to 60% and a temperature of 22 ± 2 °C. Water ad libitum and standard rodent chow were provided to the mice. Injections were given intraperitoneally (i.p.) according to the treatment group assigned. The IACUC committee at King Saud University approved all protocols and procedures for the animal study (KSU-SE-22-53).An elevated plus-shaped maze with a 45 × 60 cm Plexiglass box was used to assess animal performance. The maze was monitored by a camera connected to a computer, with the ANY-maze software (version 6) used for recordings. Open field boxes  connected to activity monitoring software  were also used to assess the distance traveled by each mouse. Other equipment included transfer chambers, an imaging system , a cryostat machine for brain dissection, and a spectrometer for protein assay.The study design and timeline are summarized in The Control group was treated with the vehicle solution  for all 17 days.The Khat group was given khat extract  for all 17 days .The Khat-CA group was given khat extract  for 17 days and treated with clavulanic acid (5 mg/kg) ,41 for tThe Khat-CA-DHK group was given khat extract  for 17 days and treated with clavulanic acid (5 mg/kg) for the last seven days of the study; in addition, they were given DHK  ,58 30 miBehavioral tests were performed before the start of treatment and on the 16th and 17th days of the experimental procedure for all experimental groups.The open field activity monitoring system comprehensively assesses mouse locomotor and behavioral activity levels and is used for the assessment of locomotive impairment in animal models of neuromuscular disease. As described previously , the micThe elevated plus maze (EPM) is used to assess anxiety-related behavior in rodent models. The EPM apparatus is a “+”-shaped maze elevated above the floor with two oppositely positioned closed arms, two oppositely positioned open arms, and a central area. The rodent is monitored, and the number of entries and the time spent in each segment are measured. It is perceived that the more relaxed a rodent is, the more willing it is to explore “open” spaces and become exposed; conversely, the more stressed the animal, the more it hides in “closed” spaces .The test was blindly performed during the animal’s light cycle, and each mouse was first habituated for 15 min then monitored for 5 min. We calculated the ratio of time in closed arms by dividing the time spent in closed arms by the total time spent in closed and open arms (Equation (1)). Similarly, the ratio of time in open arms was calculated by dividing the time spent in open arms by the total time spent in closed and open arms (Equation (2)). We also considered the raw time spent in open arms and in closed arms. Animals’ tracking and monitoring were performed using ANY-maze software.The novel object recognition test (NORT) was used as described previously to characterize mouse learning and memory ,62. The Mice were euthanized approximately 24 h after the last treatment. The brains were immediately extracted and snap-frozen in a −80 °C freezer for use in immunoblotting. Subsequently, the NAc (~1 mm thickness) was dissected (freehand) using a cryostat machine maintained at −20 °C. Six random samples were selected from each group for western blotting assay. Determination of brain region boundaries was made using the Paxinos and Watson atlas .n = 6 mice per group) was homogenized in a lysis buffer containing phosphatase and protease inhibitors. Total protein was quantified using a BCA assay, and then equal amounts were loaded on 8–15% SDS-PAGE gels. After protein separation by electrophoresis, the bands were transferred to membranes using a transfer chamber. Blocking of membranes was performed using 3% non-fat milk in TBST for one hour, after which the membranes were incubated with primary antibodies overnight at 4 °C; the antibodies used were rabbit anti-GLT-1 , rabbit anti-xCT , rabbit anti-GLAST , and rabbit anti-actin . On the second day, the membranes were incubated with an anti-rabbit secondary antibody for 90 min. Western blot detection reagents were then added, and the signal was detected with an imaging system . Image J software (Java 8) was used to quantitatively measure the expression of the proteins of interest. All treatment groups were normalized to the expression of the Control group in the same gel. In order to identify significant changes in the expression of the proteins (compared to the water control group) in the NAc, the data for the water control group were expressed as 100% as performed in previous studies [Western blotting was used to quantify GLT-1, xCT, and GLAST expression in the NAc, as described in our previous work . Briefly studies ,66,67,68Gas chromatography-mass spectrometry (GC-MS) was used to identify cathine and cathinone, two of the major psychostimulant active ingredients in khat extract, as described in a previous study . Externat-tests to compare the obtained data before and after treatments for each parameter in each group. Differences between the groups in the baseline and post-treatment periods were evaluated by a one-way ANOVA followed by Tukey’s post hoc comparison test. Protein expression of GLT-1, xCT, and GLAST was first normalized by corresponding β-actin expression and then to the Control group, after which differences were evaluated using a one-way ANOVA followed by Tukey’s post hoc test. Differences in mouse weight were assessed through a two-way ANOVA. Results were deemed statistically significant when p ≤ 0.05. GraphPad Prism was used to calculate the outliers, and these outliers were excluded from the analysis.All data were analyzed using PRISM software version 6.01. Behavioral data from the locomotion, novel object recognition, and EMP tests were evaluated using paired Because the majority of GLT-1 proteins are expressed in astrocytes, we hypothesized here that GLT-1 and xCT expression in the astrocytes in the NAc are decreased and consequently led to altered glutamatergic system and impaired neurobehavioral effects in mice repeatedly exposed to khat . Future


Klebsiella pneumoniae is a significant healthcare-associated pathogen. We investigated the antimicrobial interaction pattern between zinc sulfate and antibiotics against K. pneumoniae biofilm on the phenotypic and genotypic levels.K. pneumoniae biofilm formation genes post-treatment were carried out to evaluate the effect on the phenotypic and genotypic levels, respectively.Determining the minimum biofilm inhibitory concentrations and the transcriptomic profile of Zinc enhanced the antibiofilm potentials of cephalosporins, aminoglycosides, and ertapenem, whereas it antagonizes the effectiveness of fluoroquinolones and meropenem on the phenotypic level. On the molecular level, zinc enhanced the anti-biofilm efficacies of cephalosporins  via down-regulating the expression of biofilm-related genes by 18-, 38-, 5-, 77- and 2-folds, respectively. Zinc in combination with aminoglycosides  reduced the expression of biofilm-related genes by 40-, 2602- and 20-folds, respectively, and by 2-folds in combination with ertapenem. However, a reduction in the down-regulatory potentials of fluoroquinolones was recorded following combination with zinc by 2-, 2-, 15- and 14-folds, respectively, and an up-regulation in the expression levels of the tested genes by 2-folds in the case of zinc/meropenem combination.Results revealed variable interaction patterns between different antibiotics in combination with zinc. Current findings also shed light on the antibiofilm potentials of zinc/antibiotics combinations especially when combining zinc with fluoroquinolones or meropenem to avoid their antagonistic effects. Klebsiella pneumoniae has been categorized as a crucial Gram-negative, encapsulated, non-motile Enterobacteriaceae. K. pneumoniae typically colonizes multiple sites from where it can reach the systemic circulation resulting in severe infections either in the surgical sites or in the urinary tract. It plays a key role in ventilator-associated pneumonia as well as in hospital-acquired pneumonia. K. pneumoniae infections are also of special concern, especially in the case of immunocompromised neuropathic and diabetic patients [Among various biofilm-forming pathogens, patients .K. pneumoniae such as the presence of capsular polysaccharides, outer lipopolysaccharide, and outer membrane proteins in addition to type-3 fimbriae, where the microorganism relies on these factors to survive and to escape from the immune mechanisms during infection and development of bacterial biofilm [K. pneumoniae is of critical importance as it enhances its resistance to several antibiotics. Moreover, the rapid spread of the biofilm-forming multidrug-resistant (MDR) K. pneumoniae was demonstrated to be associated with an increased incidence of nosocomial infections leading to huge records of morbidity and mortality [K. pneumoniae is also associated with the absence of a rationale use of antibiotics [Many factors contribute to the virulence of  biofilm . The bioibiotics .Biofilms are composed of polymeric matrix enclosing bacterial communities which are characterized by their high resistance to antibiotics . This elK. pneumoniae virulence genes were found to be also engaged in biofilm formation. Examples of these genes are cps and mrk genes which are involved in capsule formation and production of type 3 fimbriae, respectively. In addition to wbbm and wzm cluster genes which contribute to the synthesis of O-antigen that is present in the bacterial lipopolysaccharide and consequently in the production of K. pneumoniae biofilms. It was also demonstrated both luxS gene which is responsible for the regulation of type-2 quorum sensing and pgaABCD operon which synthesize and translocate poly-β-1,6-n-acetyl-d-glucosamine adhesion are contributors to biofilm production via stimulating the intracellular communication and enhancing the cellular binding to abiotic surfaces, respectively [It is worth noting that ectively .K. pneumoniae. The capabilities of such antibiotics either alone or in combination with zinc to modulate the transcriptomic profile of K. pneumoniae biofilm-related-genes  were also investigated.Owing to the urgent need for alternative antimicrobial options, the application of combined therapy has attracted the attention of many scientists and clinicians as an effective strategy to overcome infections associated with biofilms. This approach might be addressed via the combination between conventional antibiotics and zinc as one of the trace elements which is essential for many vital processes  and withK. pneumoniae clinical isolate (BKP-122) was employed as a bacterial model in the current study [K. pneumoniae was streaked on tryptic soy agar (TSA) plates and further incubated for 24 h at 37 °C. Ensuring the ability of the selected isolate to produce biofilm was carried out according to Vuotto et al. using the crystal violet assay method [Previously identified biofilm-forming nt study  and it wy method , aliquot8 colony forming unit (CFU)/ml). In 96-well microtiter plates, antibiotics, as well as zinc sulfate, were two-fold serially diluted to reach a final concentration range of 0.125–8 mg/ml using CAMHB. Plates were inoculated with K. pneumoniae suspension at a final count of 5x105 CFU/ml. Positive control wells were left free from the tested agents, while other wells without bacterial inoculums were considered negative controls. MIC was regarded as the minimum concentration of each of the tested agents that exhibited no bacterial growth following 18 h incubation at 37 °C using resazurin indicator, where the color change of the resazurin from purple to pink or colorless indicates bacterial growth [The minimum inhibitory concentrations of the tested antibiotics as well as zinc sulfate were determined using the broth microdilution method . The bacl growth .Bacterial inoculum matching 0.5 MacFarland was diluted 100 times using LB broth and inoculated (200 µl/well) in microtiter plates. Negative control wells were inoculated with the medium only. The ability of the bacterial isolate to form biofilm following 18 h incubation was previously ensured using crystal violet assay method . Plates K. pneumoniae biofilm formation genes  were determined using real-time PCR (Table K. pneumoniae to reach the mid-log phase (18 h). The bacterial suspension was further diluted in LB broth (supplemented with 1% glucose) to an OD600 of 0.01 and inoculated in 12-well flat-bottomed cell culture plates.The existence and the transcriptomic profile of CR Table . Bacteri4 (0.25 MIC—1 mg/ml), while other wells were treated with the same concentration of the tested antibiotic only. In the meantime, the effect of ZnSO4 (0.25 MIC) was examined by adding it alone to the corresponding wells at a final concentration of 1 mg/ml, whereas positive control wells were left untreated. Plates were incubated for 18 h at 37 °C under static conditions to allow biofilm formation. The contents of all wells were gently aspirated and rinsed two times with phosphate buffer saline and the formed biofilms were detached from the plate’s surface and collected by centrifugation. A lysis solution was used to pre-treat the collected biofilms. RNA was extracted from treated cells during the biofilm development phases in addition to the positive control cells using Gene JET RNA Purification Kit  as recommended by the producer.Plates were inoculated with 0.5 MBIC of each antibiotic in combination with ZnSOUtilizing a NanoDrop spectrophotometer, the RNA yield was measured. According to the manufacturer’s guidelines, DNA contamination was eliminated using DNaseI . The purified RNA was reverse transcripted into cDNA and amplified using TOPreal one-step RT qPCR Kit—SYBR Green with low ROX . Normalization of the expressed levels of mRNA was performed using the 23S rRNA gene as an internal control gene. Real time-PCR (RT-PCR) was performed using 1 µl of diluted RNA (10 ng/ µl) for each gene, along with 10 µl of TOPreal one-step RT qPCR Reaction Mix and 1 µl of TOPreal one-step RT qPCR Enzyme Mix in addition to 1 µl of each primer (10 pmol/µl) in a final volume of 25 µl reaction. All reagents except for the diluted RNA were utilized for the negative controls.23S rRNA).The 2-∆∆cycle threshold  and tested antibiotics (0.5 MBIC) alone and in combinations. The bacterial growth was evaluated turbidimetrically in independent triplicates by measuring the OD600 using ELx800, Biotek  at 2, 4, 6, 18, 20 and 24 h time intervals and compared to the control wells [The bacterial growth rate was determined in order to evaluate the growth inhibitory potentials of the tested agents at the tested concentrations to exclude any possible effect on biofilm due to reduced bacterial growth. Briefly, an 18 h bacterial culture was adjusted to an ODol wells .The data acquired by the quantitative biofilm formation experiment were analyzed using the student’s t-test using GraphPad Prism software . The differences were regarded as statistically significant at P < 0.05, and the confidence level was 95%.K. pneumoniae isolate demonstrated antibiotic resistance to ceftazidime, ceftriaxone, cefotaxime, cefepime, kanamycin, and gentamicin in addition to intermediate sensitivity to ofloxacin. Whereas it was sensitive to gatifloxacin, ciprofloxacin, amikacin, and meropenem. Recorded data showed that among the four tested classes of antimicrobial agents, the selected K. pneumoniae isolate was resistant to at least one antibiotic from three different antibiotic classes , thus it was considered multidrug-resistant  potentiates the antibiofilm effectiveness of ceftazidime, ceftriaxone, cefotaxime, cefepime, and cefpirome, where the MBIC decreased by a value of 16-, 8-, 4-, 4- and 8-folds, respectively. In the meantime, an enhancement in the antibiofilm potentials of kanamycin, gentamicin, amikacin, and ertapenem was recorded following combination with zinc sulfate in the order of 4-, 2-, 4- and 8-folds, respectively. On the other side, a reduction in the antibiofilm efficacies was observed following the combination between zinc sulfate and gatifloxacin, ciprofloxacin, moxifloxacin, ofloxacin, and meropenem by 4-, 4-, 2-, 2- and 4-folds, respectively. One dilution below the recorded MBIC of each of the tested antibiotics either in the presence or in the absence of zinc sulfate, where it confirms the biofilm formation (sub-MBIC), was further applied to examine the transcriptomic profile of biofilm formation genes as indicated in Table K. pneumoniae biofilm was assessed by comparing the expression profile of genes controlling biofilm formation using RT-PCR. Current findings demonstrated that zinc sulfate alone reduced the expression of biofilm-correlated genes luxS, mrkA, pgaA, wbbM, and wzm genes by 2.8-, 4.1-, 7.6-, 1.5- and 2.4-folds, respectively as compared to the control bacterial cells  alone and in combination with zinc sulfate 0.25 MIC—1 mg/ml) against  mg/ml agK. pneumoniae biofilm-associated genes, it was found that the expression levels of luxS, mrkA, pgaA, wbbM, and wzm genes were down-regulated by 16.5-, 21-, 18-, 7- and 35.4-folds as compared to that observed following exposure to cefotaxime alone, respectively. Also, zinc sulfate/ceftriaxone combination reduced the expression of luxS, mrkA, pgaA, wbbM, and wzm genes by 2.7-, 79.3-, 105-, 4.3-, and 1.7-folds, respectively. Moreover, the expression levels of the tested genes were decreased in the order of 9.9-, 6.9-, 6.1-, 5.9- and 9.3-folds following treatment with zinc sulfate and ceftazidime in combination, respectively. Analogously, zinc sulfate/cefpirome combination down-regulated the expression of luxS, mrkA, pgaA, wbbM, and wzm genes by 324.7-, 68.8-, 11.9-, 1.7- and 3.7-folds, respectively. Furthermore, the expression levels of luxS and wbbM genes were reduced by values of 4.3-, and 5.2-folds post-exposure to zinc sulfate/cefepime combination compared to that observed following treatment with cefepime alone, respectively  via down-regulating the expression of biofilm-related genes in the order of 18-, 38-, 5-, 77- and 2-folds, respectively. Concerning the effect of zinc sulfate/cefotaxime combination on luxS, mrkA, pgaA, wbbM, and wzm genes by 32.7-, 68.8-, 116.9-, 1.7- and 3.7-folds, respectively as compared to that with kanamycin alone. Moreover, zinc sulfate/gentamicin combination reduced the expression of luxS, mrkA, pgaA, wbbM, and wzm genes in the order of 3548.3-, 1214.2-, 785.5-, 7541.3- and 163.1-folds, respectively as compared to that with gentamicin alone. Additionally, zinc sulfate/amikacin combination exhibited down-regulation of the expression levels of luxS, mrkA, pgaA, wbbM, and wzm genes by values of 0.12-, 32.7-, 65.3-, 1.5- and 3.2-folds as compared to that with amikacin alone, respectively , results revealed that zinc sulfate in combination with the tested aminoglycosides could reduce the expression of biofilm-elated genes by 40-, 2602- and 20-folds, respectively. The recorded data showed that zinc sulfate/kanamycin combination down-regulated the expression levels of luxS, mrkA, pgaA, wbbM, and wzm genes by 0.057–0.0025-, 0.00016-, 0.0038- and 0.08-folds compared to that observed in case of moxifloxacin, respectively. A comparable pattern was detected in the case of zinc sulfate/ofloxacin combination, where the expression levels of the biofilm-related genes were reduced in the order of -0.42-, 0.00009-, 0.01-, 1.71- and -0.0035-folds compared to ofloxacin, respectively. Moreover, the down-regulation of the tested genes was decreased following the combination between zinc sulfate and ciprofloxacin by values of 0.053-, 0.0086-, 0.00033-, 0.00044- and 0.0072-folds, respectively, compared to that recorded post ciprofloxacin treatment. In the same context, zinc sulfate antagonizes the efficacy of gatifloxacin by reducing the expression of luxS, mrkA, pgaA, wbbM, and wzm genes by 0.26-, 1.247-, 0.223-, 0.000001- and 0.0068-folds as compared to that observed in case of gatifloxacin alone, respectively  in combination with zinc sulfate showed an antagonistic effect. That was apparent in that the tested antibiotics alone showed down-regulatory potentials on the biofilm-related genes, however, the combination of these antibiotics with zinc sulfate exhibited a reduction in this down-regulation by 2-, 2-, 15- and 14-folds, respectively. Recorded results demonstrated that zinc sulfate in combination with moxifloxacin antagonizes the antibiofilm potentials of moxifloxacin via reducing the down-regulation of the expression levels of luxS, mrkA, pgaA, wbbM, and wzm genes by 5.1-, 1.7-, 3.2-, 8.1- and 0.7-folds, respectively. On the other hand, it antagonizes the effectiveness of meropenem by up-regulating the expression of luxS, mrkA, pgaA, wbbM, and wzm genes by 1.2- 1.7-, 0.0003-, 0.009- and 1.9-folds, respectively  is MDR. MDR was based on resistance to at least one antibiotic from three different antibiotic classes, namely cephalosporins, aminoglycosides, and carbapenems. Additionally, 0.25 MIC of zinc sulfate (1 mg/ml) was applied in the present study, where this concentration of zinc sulfate has a negligible effect on planktonic cells and consequently its antibiofilm potential on the tested bacterial isolate would be null. A comparable concentration of zinc sulfate was also applied in another study to investigate the effect of zinc in combination with different antibiotics against Pseudomonas aeruginosa biofilms [l wounds . Antibiol wounds . Consequbiofilms .K. pneumoniae, where the isolates capable of biofilm formation were found to be MDR compared to non-biofilm producers. That was probably correlated to that the bacterial plasmids carrying antibiotic-resistance genes could also express genes participating in bacterial adherence as well as biofilm formation [Biofilm eradication is challenging, where the existence of the metabolically inactive persister cells in the matrix of the bacterial biofilm renders these bacteria unable to be destroyed by antimicrobials . This juormation .n-acetylglucosamine (PGA). On the other side, the ability of zinc to inhibit the bacterial biofilms of other microorganisms that lack PGA in their biofilms indicated that it is not the only inhibitory mechanism [E. coli and K. pneumoniae biofilms by hindering iron uptake which is essential in biofilm formation [K. pneumoniae biofilm via the down-regulation of biofilm-related genes. In the mean context, the tested cephalosporins as well as aminoglycosides in addition to ertapenem in combination with zinc down-regulated the expression levels of biofilm-associated genes.Despite that, the antibiofilm effectiveness of zinc has not been fully recognized but zinc may exert selective pressure on bacteria that can produce biofilm via different mechanisms. Zinc could react with the constituents of the bacterial biofilm such as poly-echanism . Anotherechanism . It was ormation . Zinc waormation . In addiRegarding the ability of zinc to enhance the antibacterial effectiveness of ceftazidime as an example of the third-generation cephalosporins, it was demonstrated that three active groups were responsible for the antibacterial activity of ceftazidime. It was reported that the increased affinity to penicillin-binding proteins of Gram-negative bacteria is brought on by the aminothiazole ring as well as the propyl-carboxy group at position 7 of ceftazidime. The pyridine group in position 3 of the ceftazidime molecule, which promotes intra-bacterial penetration in a short time, is another active moiety. It was found that the formation of a stable Zn-antibiotic complex by the chelation of Zn with the propyl-carboxy group (at position 7) of ceftazidime could facilitate its entrance into the bacterial cells and consequently enhance its antimicrobial potential .E. coli due to the chelation interaction between zinc and ciprofloxacin. On the other hand, gentamicin as a non-fluoroquinolone antibiotic that cannot chelate zinc ions did not demonstrate a comparable effect in presence of zinc ions due to the lack of zinc-gentamicin interaction [In this study, the reduced antibiofilm activity of fluoroquinolones in combination with zinc but not in the case of aminoglycosides could be attributed to the ability of fluoroquinolones to chelate zinc ions. A study reported that zinc combination with ciprofloxacin reduced the effectiveness of ciprofloxacin against eraction . It was eraction .K. pneumoniae biofilm-related genes by a reduced level as compared to that in the case of fluoroquinolones alone. It was reported that the coexistence of two stress factors as antibiotics in addition to metal ions could play an alarming role in the de novo development of resistance to antibiotics, especially in the complicated bacterial populations where there is an elevated chance for increased rates of mutation and the transfer of resistance genes horizontally [On the molecular level, current findings demonstrated consistency with the results on the phenotypic level where zinc in combination with fluoroquinolones down-regulated zontally . Furtherzontally .The CLSI reported that Enterobacteriaceae which produce carbapenemase are usually showing intermediate susceptibility or resistance to one or more carbapenem as well as resistance to one or more of the third-generation cephalosporins considering that the resistance to ertapenem highly indicates the production of carbapenemase . Thus, cK. pneumoniae is characterized by its ability to produce metallo-b-lactamases (MBLs) which play a key role in the resistance to carbapenems although other mechanisms are involved in this resistance. MBLs are transmissible carbapenemases that depend on zinc ions in their activity. A recent study reported that zinc chelators could restore the antibacterial activity of meropenem against K. pneumoniae by acting as an inhibitor to MBLs [ to MBLs . That coK. pneumoniae strain showed sensitivity to meropenem and on the contrary, it was resistant to ertapenem. Such finding could indicate that ertapenem might be inactivated by a resistance mechanism which was not applicable in the case of meropenem as there was no resistance against it in the current study particularly since our results on the molecular level were coherent with that on the phenotypic level. In the meantime, evaluating the bacterial viability following different treatments indicated that the recorded difference in gene expression is not due to the effect of different treatments on the bacterial growth rate.On the other side, the detected synergism in the case of ertapenem in combination with zinc could be related to the demonstrated variability in the affinity of zinc to different MBLs , taking K. pneumoniae biofilm formation genes. The current findings are of critical interest, especially in the case of the reported antagonism between some antibiotics in combination with zinc against K. pneumoniae biofilm. This issue will be more problematic in patients who receive zinc as a supplement [To the best of our knowledge, this is the first report demonstrating the modulatory potentials of zinc sulfate in combination with different antibiotic classes on the transcriptomic profile of MDR pplement  as it coK. pneumoniae biofilm on both the phenotypic and genotypic levels with a potential enhancement of the antibiofilm effectiveness in the case of cephalosporins, aminoglycosides as well as ertapenem. On the other hand, a significant antagonism was detected following the combination of zinc sulfate with fluoroquinolones and meropenem.The present study revealed a variable range of the modulatory potentials of zinc sulfate in combination with different antibiotics against MDR


Bifidobacterium bifidum G9-1 (BBG9-1) on the intestinal environment and clinical features in patients with qCD. Eleven patients with qCD, who met the Rome III diagnostic criteria for IBS-D, received BBG9-1 (24 mg) orally three times daily for 4 weeks. Indices of the intestinal environment  and clinical features  were evaluated before and after treatment. Treatment with BBG9-1 tended to reduce the IBS severity index in the studied patients (p = 0.07). Among gastrointestinal symptoms, abdominal pain and dyspepsia tended to be improved by the BBG9-1 treatment , and IBD-related QOL showed a significant improvement (p = 0.007). With regard to mental status, the patient anxiety score was significantly lower at the endpoint of BBG9-1 treatment than at the baseline (p = 0.03). Although BBG9-1 treatment did not affect the fecal calprotectin level, it suppressed the serum MCP-1 level significantly and increased the abundance of intestinal Bacteroides in the study patients. The probiotic BBG9-1 is able to improve IBD-related QOL with a reduction of anxiety score in patients with quiescent CD and IBS-D-like symptoms.Diarrhea-predominant irritable bowel syndrome (IBS-D)-like symptoms are distressing for patients with quiescent Crohn’s disease (qCD) and worsen their quality of life. In the present study, we assessed the effect of the probiotic  Although the etiology of Crohn’s disease (CD) is still unclear, treatment for CD patients has greatly improved due to the development of biologic agents, resulting in a high rate of quiescent remission . AlthougBifidobacterium bifidum G9-1 (BBG9-1) on the intestinal environment and clinical features in patents with qCD.It is noteworthy that a large group 30–50%) of patients with qCD have IBS-like symptoms ,3,4,7 al0–50% of Patients who had qCD with IBS-D-like symptoms were enrolled into this prospective intervention study at our university hospital between March and October 2021. The protocol was designed according to the Declaration of Helsinki, approved by the Japan Physicians Association Institutional Review Board  and registered in the Japan Registry of Clinical Trials (jRCT) (Registration No. jRCTs 031200327). Similarly to previous work ,10,13,14Patients who met all of the conclusion criteria for both qCD and IBS-D were enrolled. Clinical data and symptoms were assessed by a self-completed symptom questionnaire. Patients satisfying the inclusion and exclusion criteria  were regThis prospective open label intervention study comprised a 4-week period of treatment with the probiotic BBG9-1 . Twelve The primary endpoint was the change in the fecal calprotectin level at the end of the treatment relative to that at the start. The secondary endpoint was the changes in serum inflammation markers and IBD/IBS-related activity, symptoms and QOL at the end of the treatment relative to the values at the start.See “Materials and Methods” in reference  and assoSee “Materials and Methods” in reference .See “Materials and Methods” in reference .Microbiome diversity was assessed using the Shannon index, Faith PD (phylogenetic diversity), and observed OTUs  based on 97% nucleotide sequence identity. Comparison of each taxon in the gut microbiota was performed at the genus level.t test was applied to compare clinical data, questionnaire scores, serum cytokines level, fecal calprotectin level and microbiota diversity. Wilcoxon signed-rank test was applied for comparison of microbiome abundance. Statistical significance was p < 0.05.All statistical analyses were carried out using SAS version 9.4 . Results are shown as means ± SD or median and inter-quartile range. Paired p = 0.07) from 170 [60–240] to 110 [80–220]  relative to that at the baseline , but not to a significant degree. The IBS severity index was decreased almost significantly ([80–220] .p = 0.09). The observed improvements in abdominal pain and dyspepsia reached an almost significant level .Gastrointestinal symptoms including reflux, abdominal pain, dyspepsia, diarrhea and constipation was evaluated using a gastrointestinal symptom rating scale . Overallp = 0.05).We next assessed the effect of the probiotic BBG9-1 on the QOL of patients with qCD and IBS-D-like symptoms . The IBDUsing the HADS questionnaire, we evaluated in more detail the mental status of patients with qCD and IBS-D-like symptoms . AlthougTo assess the change in the inflammatory condition of the intestinal environment in patients with qCD and IBS-D-like symptoms, we measured the level of fecal calprotectin . This reWe also examined the levels of serum cytokines as inflammatory markers in the study patients. As shown in p = 0.08)  A. The Sh = 0.08) A.p = 0.049).Furthermore, we investigated the gut microbiome profile at the genus level before and after BBG9-1 treatment in the study patients B. We fouBifidobacterium bifidum) to patients with qCD and IBS-D-like symptoms and found that it improved their IBD-related QOL and relieved their abdominal pain. Moreover, although not to a statistically significant degree, the severity of IBS was markedly reduced by the treatment. These findings suggest that the probiotic BBG9-1 would be a promising for treatment of patients with qCD and IBS-D-like symptoms.As the number of patients with qCD has been increasing due to advances in medication, the management for those patients is gathering attentions. As IBS-D-like symptoms certainly disturb the QOL of such patients ,26, it iIt still remains unclear how BBG9-1 ameliorates IBS-D-like symptoms and improves QOL. In general, it is well known that clinical symptoms and QOL is likely affected by placebo effect . On the Bacteroides was significantly increased after the treatment with BBG9-1. Interestingly, Bacteroides has been reported to influence depression and anxiety by modulating the function of GABA [In this study, we showed that BBG9-1 treatment greatly improved the QOL of patients with qCD and IBS-D-like symptoms in term of psychological anxiety. It is well known that mental stress plays a key role in the pathophysiology of IBS . Recent  of GABA . Moreove of GABA . AlthougBifidobacterium but also Bacteroides strains play a role in the reduction of serum cholesterol level [We have additionally reported interesting findings that serum level of total cholesterol and HDL was significantly decreased in qCD patients with IBS-D-like symptoms after BBG9-1 treatment . There iol level ,46. The ol level ,47. Impool level ,42. Furtol level ,48. Togeol level ,50. FromBacteroides, thus ameliorating psychological stress. Since psychological stress is a key factor that can worsen abdominal symptoms in IBS patients, the observed improvement of IBD-related QOL by BBG9-1 may have been be due to relief of anxiety as a result of the increase in beneficial strains. However, it is known that a placebo can also greatly affect abdominal symptoms in patients with IBS [In summary, we have demonstrated that the probiotic BBG9-1 is effective for improvement of IBD-related QOL in patients with qCD and IBS-D-like symptoms, although it did not affect minimal intestinal inflammation, at least in terms of the fecal calprotectin level. This suggests that the observed improvement of IBS-D-like symptoms in those patients was not associated with amelioration of minimal intestinal inflammation. We also clarified that BBG9-1 was effective for reducing anxiety and increasing the abundance of with IBS . As we dwith IBS . Althoug